Nucleoside analogues

ABSTRACT

Nucleoside analogues containing the degenerate base analogue P and derivatives thereof are provided with reporter moieties preferably comprising signal moieties. The nucleoside analogues are useful for labeling DNA or RNA or for incorporating in oligonucleotides.

CROSS REFERENCE TO RELATED APPLICATIONS

This is the national phase of PCT/GB97/00312 filed Feb. 3, 1997 whichwas based upon Great Britain application Ser. No. GB 9602025.0 filedFeb. 1, 1996.

Nucleic acids are manipulated in vitro in a wide variety of research anddiagnostic techniques. The methods can involve the synthesis of nucleicacid probes by means of polymerase or terminal transferase enzymes forthe purposes of labelling or determination of base sequence identity.Labelling often involves the incorporation of a nucleotide which ischemically labelled or which is of a particular chemical composition soas to make it detectable. Nucleic acid probes made in this way can beused to determine the presence of a nucleic acid target which has acomplementary sequence by means of hybridisation of the probe to thetarget.

Another method for introducing chemically labelled or otherwise modifiednucleotides into DNA involves chemical synthesis using nucleosidephosphoramidite or other precursors which are linked together in anydesired sequence in oligonucleotide synthesisers, the final productbeing indistinguishable from DNA made by the use of polymerases.

In certain situations it is useful to be able to incorporate a baseanalogue into an oligo- or poly-nucleotide which does not have the basepairing specificity of the natural bases. This invention describes newnucleoside analogues which are capable of forming base pairs with morethan one nucleotide, and which carry a reporter group.

P Kong Thoo Lin and D M Brown reported (Nucleic Acids Research, 1989,Vol 17, pages 10373-10383) the synthesis of a monomer containing thedegenerate base analogue P, (6H,8H-3-4-dihydropyrimido[4,5-c][1.2]oxazin-7-one). Due to the ability ofthis base analogue to exist in amino and imino tautomers, it can basepair with both purine bases A and G. The authors found that oligomerscontaining one or more P bases formed DNA duplexes of comparablestability to the parent duplexes and which also showed sharp transitionson melting. Further evidence confirmed that the base pairs P/A and P/Gwere essentially of the Watson-Crick type.

The authors also discussed the potential use of this base analogue inhybridisation probes and primers, when the base can be put at positionsof degeneracy thus both avoiding the need for multiple-chain primers (orprobes) and significantly reducing the chain multiplicity. Indeedoligonucleotides containing several P bases were effective in dot blothybridisation and DNA sequencing experiments.

In Nucleic Acids Research, 1992, Vol 20, No 19, pages 5149-5152, theseauthors also demonstrated the use of oligonucleotides containing the Pbase at the 3′-end and elsewhere as primers in polymerase chain reaction(PCR) experiments.

EP 0 235 301 describes pyridopyrimidine nucleotide derivatives which canform base pairs with guanine or adenine, and which are fluorescent intheir own right. Excitation of, these derivatives is in the UV region(330-350 nm)

Purine and pyrimidine base nucleosides and nucleotides have beenderivatised with reporter groups and are well known and widely used forlabelling DNA or RNA and in other molecular biology applications. Butthese molecules are capable of base-pairing only with one of A, C, G andT and the nucleoside triphosphates are often poor enzyme substrates.There is a need for a nucleoside analogue whose triphosphate is a goodenzyme substrate and which has a base analogue that is degenerate, byhaving the ability to base pair with two or three of the natural basese.g. with both pyrimidines (T/C) or both purines (A/G), or universal, byforming base-pairs with each of the natural bases withoutdiscrimination. This invention makes use of the P base to address theseneeds.

The invention provides a nucleoside analogue of the formula

X is O, S, Se, SO, CO or N—R¹⁰,

the curved dotted line represents an optional link between R⁶ and R¹⁰,

R¹, R², R³ and R⁴ are the same or different and each is H, OH, F, NH₂,N₃, O-hydrocarbyl, or a reporter moiety,

R⁵ is OH or mono-, di- or tri-phosphate or -thiophosphate orcorresponding boranophosphate,

or one of R² and R⁵ is a phosphoramidite or other group forincorporation in a polynucleotide chain,

Z is O, S, Se, SO, NR⁹ or CH₂,

and R⁶, R⁷, R⁸, R⁹ and R¹⁰ are the same or different and each is H oralkyl or aryl or a reporter moiety,

n is 0 or 1,

provided that at least one reporter moiety is present,

wherein a reporter moiety comprises a linker group, together with asignal moiety or a solid surface or a reactive group by which a signalmoiety or a solid surface may be attached to the nucleoside analogue.

A nucleoside analogue is a molecule which is capable of beingincorporated, either chemically or enzymatically, into an oligomeric orpolymeric nucleic acid (DNA or RNA) chain, and when so incorporated offorming a base pair with a nucleotide in a complementary chain or basestacking in the appropriate nucleic acid chain.

A reporter moiety may be any one of various things. It may be aradioisotope by means of which the nucleoside analogue is renderedeasily detectable, for example 32-P or 33-P or 35-S incorporated in aphosphate or thiophosphate or phosphoramidite or H-phosphonate group, oralternatively 3-H or 125-I. It may be a stable isotope detectable bymass spectrometry. It may be a signal moiety e.g. an enzyme, hapten,fluorophore, chemiluminescent group, Raman label or electrochemicallabel. The reporter moiety may comprise a signal moiety and a linkergroup joining it to the remainder of the molecule, which linker groupmay be a chain of up to 30 carbon, nitrogen, oxygen and sulphur atoms,rigid or flexible, unsaturated or saturated as well known in the field.The reporter moiety may comprise a solid surface and a linker groupjoining it to the rest of the molecule. The reporter moiety may consistof a linker group with a terminal or other reactive group, e.g. NH₂, OH,COOH, CONH₂ or SH, by which a signal moiety and/or a solid surface maybe attached, before or after incorporation of the nucleoside analogue ina nucleic acid chain. Such reporter groups are well known and welldescribed in the literature.

R¹, R², R³ and R⁴ may each be H, OH, F, NH₂, N₃, O-alkyl or a reportermoiety. Thus ribonucleosides, and deoxyribonucleosides anddideoxyribonucleosides are envisaged together with other nucleosideanalogues. These sugar substituents may contain a reporter moiety inaddition to the one or two present in the base.

R⁵ is OH or mono-, di- or tri-phosphate or -thiophosphate orcorresponding boranophosphate. Alternatively, one of R² and R⁵ may be aphosphoramidite or H-phosphonate or methylphosphonate orphosphorothioate or an appropriate linkage to a solid surface e.g.hemisuccinate controlled pore glass, or other group for incorporation,generally by chemical means, in a polynucleotide chain. The use ofphosphoramidites and related derivatives in synthesisingofigonucleotides is well known and described in the literature. Fromnucleosides (R⁵ is OH) it is readily possible to make the correspondingnucleotides (R⁵ is triphosphate) by literature methods.

In the new nucleoside analogues to which this invention is directed, atleast one reporter moiety is present preferably in the base analogue orin the sugar moiety or a phosphate group. Reporter moieties may beintroduced into the sugar moiety of a nucleoside analogue by literaturemethods (e.g J. Chem. Soc. Chem. Commun. 1990, 1547-8; J. Med. Chem.,1988, 31. 2040-8). Reporters in the form of isotopic labels may beintroduced into phosphate groups by literature methods (AnalyticalBiochemistry, 214, 338-340, 1993; WO 95/15395).

The base analogue with which this invention is concerned is a fused ringstructure in which one ring contains an optional double bond shown inthe diagram as a dashed line. When a double bond is present, the ring isflat and no chirality problem arises. When a single bond is present, theintroduction of a reporter moiety R⁶ or R⁸ creates a chiral centre. Itmay be helpful if the chirality is chosen so that the reporter sticksout of, and not back into, the nucleic acid helix.

The nucleoside analogues of this invention are useful for labelling DNAor RNA or for incorporating in oligonucleotides, with the advantage overconventional hapten labelled nucleotides such as fluorescein-dUTP ofbeing able to replace more than one base or more efficient enzymaticincorporation. A reporter moiety is attached at a position so as not tointerfere with the physical or biochemical properties of the nucleosideanalogue, in particular its ability to be incorporated in singlestranded and double stranded nucleic acids without significantlyreducing the Tm. A template containing the incorporated nucleosideanalogue of this invention is suitable for copying in nucleic acidsynthesis. If a reporter moiety of the incorporated nucleoside analogueconsists of a linker group, then a signal moiety can be introduced intothe incorporated nucleoside analogue by being attached through aterminal reactive group of the linker group.

In the nucleoside analogue described above, a deoxy or dideoxy or othersubstituted ribose moiety is linked at the 1′-position to a baseanalogue. This base analogue is preferably capable of acting as a haptenso as to bind an antibody and of doing this whether or not a reportermoiety is present. In another aspect the invention provides a method ofdetecting a nucleoside analogue, as described herein but in which areporter moiety may or may not be present, said nucleoside analoguebeing incorporated in a single stranded or double stranded nucleic acidchain, which method comprises using for detection an antibody whichbinds to the base analogue thereof.

In primer walking sequencing, a primer/template complex is extended witha polymerase and chain terminated to generate a nested set of fragmentswhere the sequence is read after electrophoresis and detection(radioactive or fluorescent). A second primer is then synthesised usingthe sequence information near to the end of the sequence obtained fromthe first primer. This second (“walking”) primer is then used forsequencing the same template. Primer walking sequencing is moreefficient in terms of generating less redundant sequence informationthan the alternative “shot gun” approach.

The main disadvantage with primer walking is the need to synthesise awalking primer after each round of sequencing. Cycle sequencing requiresprimers that have annealing temperatures near to the optimal temperaturefor the polymerase used for the cycle sequencing. Primers between 18 and24 residues long are generally used for cycle sequencing. The size of apresynthesised walking primer set required has made primer walking cyclesequencing an impractical proposition. The use of nucleoside analoguesthat are degenerate or universal addresses this problem. The use of suchanalogues that are also labelled, e.g. the nucleoside analogues of thisinvention will also help to overcome the problem. Preferred reportermoieties for this purpose are radioactive isotopes or fluorescentgroups, such as are used in conventional cycle sequencing reactions.

The nucleoside analogues of this invention can also be used in any ofthe existing applications which use native nucleic acid probes labelledwith haptens or fluorophores, or other reporter groups, for example onSouthern blots, dot blots and in polyacrylamide or agarose gel basedmethods. The probes may be detected with antibodies targeted eitheragainst haptens which are attached to the base analogues or against thebase analogues themselves which would be advantageous in avoidingadditional chemical modification. Antibodies used in this way arenormally labelled with a detectable group such as a fluorophore or anenzyme. Fluorescent detection may also be used if the base analogueitself is fluorescent or if there is a fluorophore attached to thenucleoside analogue.

The nucleoside analogues of the present invention with the combinationof molecular diversity and increased numbers of positions where reportergroups may be added can result in a series of improved enzymesubstrates.

The nucleoside analogue of the present invention in its imino and aminotautomeric form pairing with adenine and guanine is shown below. In eachcase C₁ represents a deoxy or dideoxy or other substituted ribosemoiety.

DETAILED DESCRIPTION OF THE INVENTION

5-Allyl-2′-deoxyuridine is the initial building block for the synthesisof the range of P base analogues (X=O) described in the followingexamples. The synthesis of 5-allyl-2′-deoxyuridine is described by J. L.Ruth and D. E. Bergstrom J. Org. Chem. 43, 2870-2876 (1978) along withthe corresponding 5-allyl-uridine derivative. To those skilled in theart of chemical synthesis it is obvious that, by using suitableprotecting groups on the 5-allyl-uridine ribose sugar, related ribosederivatives of example compounds (2.4), (3.6), (3.7),(4.4) and theirrelated triphosphate derivatives can be readily synthesised. Such aprotecting group could be tert-butyldimethylsilane thus maintaining thesame silyl protecting group strategy used throughout the describedsynthesis. In example 6 there are described methods for the generationof a protected 5-(2-chloroethyl)-uridine that could be used to provideribose derivative analogues of example compounds (6.4), (7.3), (7.4) and(8.5).

Similarly, to one skilled in the art of chemical synthesis, it isobvious that ribo or deoxy ribo compounds described within the followingexamples could easily be converted to the dideoxy derivatives by knownmethods.

EXAMPLE 1 Synthesis of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-(carbonylmethyl)-2′-deoxyuridine(1,3) and3′,5′-O-(1l,3-(1,3,3-tetraisopropyldisiloxanylidene))-5-(2,3-epoxypropyl)-2′-deoxyuridine(1,4)

Preparation of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-(2-propenyl)-2′-deoxyuridine(1.1)

5-Allyl-2′-deoxyuridine was prepared according to literature procedures;G. B. Dreyer & P. B. Dervan, Proc. Natl. Acad. Sci. USA, 82, 968-972,(1985); J. L. Ruth & D. E. Bergstrom, J. Org. Chem., 43, 2870, (1978).

5-Allyl-2′-deoxyuridine (11.3 g, 42mmol) was first dried by twicedissolving in dry pyridine and evaporating to dryness under reducedpressure. The resulting foam was redissolved in dry pyridine (50ml),placed under nitrogen atmosphere and cooled to 0° C. in an ice-waterbath. To this solution was added1,3-dichloro-1,1,3,3-tetraisopropyldisiloxane (12.6 g, 42 mmol), viasyringe over 5 minutes. The resulting mixture was stirred for 16 hoursat room temperature, then the solvent was removed by evaporation underreduced pressure. The residue was partitioned between ethyl acetate andwater; the organic layer was retained, washed with water and brine, thendried (Na₂SO₄), filtered and evaporated under reduced pressure to asticky solid. This was purified by flash column chromatography (silica;ethyl acetate 50%: light petroleum 50%) to give the title compound (1.1)14.5 g (68%). Mpt. 168° C.; UV (CHCl₃) 268 nm. δ_(H)(300 MHz; CDCl₃)0.85-1.05 (28H, m, Si—iPr×4), 2.18-2.48 (2H, m, sugar 2′), 3.02 (2H, m,—CH ₂—CH═CH₂), 3.72 (1H, m, sugar 4′), 3.99 (2H, m, sugar 5′), 4.45 (1H,m, sugar 3′), 5.78 (1H, m, 5.06 (2H, m, —CH₂—CH═CH₂), 5.78 (1H, m,—CH₂—CH═CH₂), 6.02 (1H, m, sugar 1′), 7.24 (1H, s, C6), 8.44 (1H, s, N3)ppm

Preparation of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-(2(R,S),3-dihydroxypropyl)-2′-deoxyuridine(1.2)

3′,5′-O-(1,3-(1,1,3,3-tetraisopropyldisiloxanylidene))-5-(2-propenyl)-2′-deoxyuridine(1.1) (7.0 g, 13.7mmol) and N-methylmorpholine-N-oxide (4.79. 40 mmol)were dissolved in acetone (200 ml). To this solution was added asolution of potassium osmate dihydrate (50 mg) in water (10 ml),dropwise over 5 minutes. The resulting mixture was stirred at roomtemperature for 16 hours, then the solvent was removed by evaporation.The residue was partitioned between diethyl ether and water; the organiclayer was retained, washed with water and brine, then dried (Na₂SO₄),filtered and evaporated under reduced pressure. The residue was purifiedby flash column chromatography (silica; 5-15% methanol/dichloromethane)to give the title compound (1.2) as a white foam, 5.4 g (72%). UV(CHCl₃) 270 nm.

δ_(H)(300 MHz; CDCl₃) 0.89-1.06 (28H, m, Si—iPr×4), 1.5-2.0 (1H, broad,OH), 2.14-2.57 (4H, m, sugar 2′+—CH ₂—CHOH—CH₂OH), 3.47-3.80 (5H, m,sugar 4′+CH₂—CHOH—CH ₂OH +OH), 3.97-4.09 (2H, m, sugar 5′), 4.46 (1H, m,sugar 3′), 6.04 (1H, m, sugar 1′), 7.44+7.47 (1H, 2s, C6 diastereomers),9.0-9.4 (1H, broad, N3) ppm.

Preparation of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-(carbonylmethyl)-2′-deoxyuridine(1.3)

3′,5′-O-(1,3-(1,1,3,3-tetraisopropyldisiloxanylidene))-5-(2(R,S),3-dihydroxypropyl)-2′-deoxyuridine(2.18 g, 4.0 mmol) was dissolved in tetrahydrofuran (50 ml), then water(40 ml) added. To this solution was added a solution of sodium periodate(0.94 g, 4.4 mmol) in water (10 ml). This mixture was stirred at roomtemperature for 2 hours, then partitioned between diethyl ether andwater. The organic layer was retained, washed with water and brine, thendried (Na₂SO₄), filtered and evaporated under reduced pressure. Theresidue was purified by flash column chromatography (silica; 4%methanol/dichloromethane) to give the title compound (1.3) as a whitefoam, 1.9 g (93%). Mpt. 187° C.; UV (CHCl₃) 268 nm.

δ_(H)(300 MHz; CDCl₃+CD₃CO₂D) 0.8-1.1 (28H, m, Si—iPr×4), 2.24-2.54 (2H,m sugar 2′), 3.39 (2H, s, —CH,—CHO), 3.76 (1H, m, sugar 4′), 4.05 (2H,m, sugar 5′), 4,45 (1H, m, sugar 3′), 6.02 (1H, m, sugar 1′), 7.56 (1H,s, C6), 8.46 (1H, s, N3), 9.70 (1H, s, CH₂—CHO) ppm.

Preparation of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-(2,3-epoxypropyl)-2′-deoxyuridine(1.4)

3′,5′-O-(1,3-(1,1,3,3-tetraisopropyldisiloxanylidene))-5-(2-propenyl)-2′-deoxyuridine(1.1) (2.04 g, 4.0 mmol) was dissolved in dichloromethane (25 ml); tothis solution was then added 3-chloroperoxybenzoic acid, 55% (1.3 g=4.1mmol mCPBA). This mixture was stirred at room temperature for 16 hours.It was then washed three times with 10% aqueous sodium carbonatesolution, then brine. It was dried (Na₂SO₄), filtered and evaporatedunder reduced pressure to give a solid, which was purified by flashcolumn chromatography (silica; 25% ethyl acetate/dichloromethane) togive the title compounds (1.4), 1.25 g (59%). UV (MeOH) 266 nm.

δ_(H)(300 MHz; CDCl₃) 0.96-1.08 (28H, m, Si—iPr×4), 2.30-2.52 (4H, m,sugar 2′+—CH ₂—CH(O)CH₂), 2.65-2.77 (2H, m, —CH₂—CH(O)CH ₂), 3.11 (1H,m, —CH₂—CH(O)CH₂), 3.75 (1H, m, sugar 4′), 4.03 (2H, m, sugar 5′), 4.50(1H, m, sugar 3′), 6.06 (1H, m, sugar 1′), 7.41+7.44 (1H, 2s, C6diastereomers), 8.57 (1H, s, N3) ppm.

EXAMPLE 2 Synthesis of6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-methyl-8H-pyrimido[4,5-c][1,2]oxazin-7-one(2.4)

Preparation of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-(2(R,S)-hydroxypropyl)-2′-deoxyuridine(2.1)

To methyl magnesium bromide (35 ml of a 1.4M solution in ether, 49 mmol)at 0° C. under nitrogen was added by dropwise addition a mixture of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-(carbonylmethyl)-2′-deoxyuridine(1.3) and3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-formyl-2′-deoxyuridine(3.95 g of a 3:1 mixture by proton NMR, approx 7 mmol*) and acetic acid(100 μl, 1.75 mmol) in dry ether (35 ml) over 15 minutes. The reactionmixture was left to stir at 0° C. under nitrogen for 30 minutes beforebeing quenched by pouring into a saturated ammonium chloride solution(approx. 200 ml). The products were extracted with ethyl acetate and theorganic phase washed with water, brine, 0.1M HCl and brine again toremove the bulk of the magnesium salts. The organic layer was removedunder reduced pressure and the resulting residue subjected to a silicagel flash column purification using a methanol/dichloromethane 5:95elution solvent. Yield 1.79 g of a gum consisting of the title compounds(2.1) and the corresponding3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene)-5-(1(R,S)-hydroxyethyl)-2′-deoxyuridine.Ratio 2.2:1 by proton NMR. Rf 0.27 and 0.31 respectively inmethanol/dichloromethane 5:95 on t.i.c.

Data for mixture of products: δ_(H)(³⁰⁰ MHz; CDCl₃) 0.9-1.1 (28H, m,SiCH(CH₃)₂) 1.20-1.22 (3H, pair of d, diastereomic CH₃), 2.25-2.6 (4H,m, CH₂ 2′ CH₂ 5), 3.8 (1H, m, H4′), 3.7-4.05 (3H, m, CHOH—CH₃ CH₂5′),4.5 (1H, m, H3′), 6.05 (1H,m, H1′), 7.41-7.46 (1H, pair of s, H6),8.43 (1H, s, NH) ppm.

* NB The formyl species was formed by over-oxidation during thepreparation of compound (1.3) and was not removed during thepurification procedure. The corresponding 5-(1-R,S)-hydroxyethyl) hasproton-NMR signals at δ1.46-1.49 (pair of d, diastereomeric CH₃) 4.65(m, CHOH—CH₃) 7.50-7.58 (pair of s, H6) ppm, as well as overlap withtitle compound signals.

Preparation of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyldisiloxanylidene))-5-(2(R,S)-phthalimido-oxypropyl)-2′-deoxyuridine(2.2)

To 3′,5′-O-(1,3-(1,1,3,3-tetraisopropyldisiloxanylidene))-5-(2(R,S)-hydroxypropyl)-2′-deoxyuridine(2.1) mixed with the 1-(R,S)-hydroxyethyl analogue (1.79 g, approx. 3.4mmol of (2.1)) in dry THF (40 ml) under nitrogen at room temperature wasadded triphenylphosphine (0.886 g, 3.4 mmol), N-hydroxyphthalimide(0.551 g, 3.4 mmol) and diethyl azodicarboxylate (0.58 ml, 3.7 mmol).There was a transient red coloration which faded to yellow. The reactionmixture was then left to stir at room temperature under nitrogen for 20hours. The solvent was removed under reduced pressure and the resultantresidue subjected to silica flash column chromatography using ether,light petroleum, dichloromethane 70:15:15 as eluant. Mixed fractionswere combined and repurified again to provide title compounds (2.2) as agum. Yield 1.07 g. Rf 0.47 and 0.55 on TLC eluant ether for the twodiastereomers.

δ_(H)(300 MHz; CDCl₃) 0.9-1.05 (28H, m, SiCH(CH₃)₂), 1.3-1.4 (3H, pairof d, diastereomeric CH₃), 2.4-2.75 (4H, m, =C—CH₂, CH₂2′), 3.75 (1H, m,H4′), 4.0 (2H, m, CH₂5′) 4.2-4.6 (2H, m, H3′ and NOCH diastereomericpair), 6.1-6.25 (1H, pair of m, diasteromeric H1′), 7.65-7.82 (5H, m,ArH, H6), 8.22-8.25 (1H, pair of s, diastereomeric NH) ppm.

Preparation of 1-(3,5-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-2-deoxy-β-D-ribofuranosyl)4-triazolo-5-(2(R,S)-phthalimido-oxypropyl)-1H-pyrimidin-2-one(2.3)

To 1,2,4-triazole (4.2 g, 60.7 mmol) dissolved in dry acetonitrile (30ml) at 0° C. was added triethylamine (10.3 ml, 73.5 mmol) followed bydropwise addition of phosphorus oxychloride (1.15 ml. 12.4 mmol)dissolved in acetonitrile (10 ml) over 10 minutes. The mixture wasallowed to stir at 0° C. under nitrogen for 10 minutes followed by 20minutes at room temperature. To the reaction mixture was added dropwise,over 20 minutes,3′,5′-O-(1,3-(1,1,3,3-tetraisopropyldisiloxanylidene))-5-(2(R,S)-phthalimido-oxypropyl)-2′-deoxyuridine(2.2) (1.07 g) dissolved in dry acetonitrile (20 ml). The reactionmixture was stirred at room temperature for 50 minutes before removingthe reaction solvent under reduced pressure. The residue was partitionedbetween dichloromethane and brine. The organic phase was separated off,dried with magnesium sulphate and concentrated to a gum under reducedpressure. Product purification was performed by silica gel flashchromatography using a gradient elution with ether, ether/ethyl acetate1:1 and finally neat ethyl acetate to give the title compounds (2.3) asa gum. Yield 0.59 g. Rf 0.24 and 0.29 in ethyl acetate on silica TLC forthe two diastereomers.

δ_(H)(300 MHz; CDCl₃) 0.9-1.2 (28H, m, SiCH(CH₃)₂), 1.4-1.52 (3H, pairof d, diastereomeric CH₃), 2.5-3.0 (3H, m, sugar H2 and H2′, 1H from═C—CH₂), 3.4-5.3 (6H, series of m, sugar H4,H3, 2×H5 and 1H from ═C—CH₂,NOCH), 6.04-6.25 (1H, pair of m, diastereomeric=CH), 8.26-8.4 (1H, pairof s, diastereomeric triazole H), 9.3-9.35 (1H, pair of s,diastereomeric pair triazole H) ppm.

Preparation of6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-methyl-8H-pyrimido[4,5-c][1,2]oxazin-7-one(2.4)

1-(3,5-O-(1,3-(1,1,3,3-Tetraisopropyidisiloxanylidene))-2-deoxy-β-D-ribofuranosyl)-4-triazolo-5-(2(R,S)-phthalimido-oxypropyl)-1H-pyrimidin-2-one(2.3) (0.59 g, 0.81 mmol) was dissolved in dry dioxan saturated withammonia (100 ml) and the solution stirred at room temperature for 18hours. The dioxan was removed under reduced pressure and the cyclisedproduct purified by silica gel flash chromatography using ether aseluant to give a colourless gum. Yield 310 mg. Rf 0.44 in ether onsilica TLC; no diastereomeric separation was observed.

The sugar was deprotected in the following manner: To the product fromabove (208 mg, 0.4 mmol) dissolved in tetrahydrofuran (5 ml) was addedtetra-n-butylammonium fluoride (435 μl of a 1.0 M solution in THF, 0.44mmol) and the mixture was stirred for 5 minutes at room temperature. Thesolvent was removed under reduced pressure and the product purified bysilica gel flash chromatography using methanol/ethyl acetate 1:9 to givethe title compounds (2.4) as a clear gum. Yield 84 mg. Rf 0.33 inmethanol/ethyl acetate 1:9 on silica TLC; no diastereomeric separationwas observed.

67 _(H)(300 MHz;CD₃OD) 1.2 (3H, d, CH₃J 6.2 Hz), 2.04-2.08 (2H, m,CH₂-), 2.18-2.27 (1H, m, sugar H2), 2.52-2.57 (1H, m, sugar H2),3.6-3.75 (3H, m, sugar 2×H5 and CH—CH₃), 3.75 (1H, m, sugar H4), 4.27(1H, m, sugar H3), 6.15 (1H, m, sugar H1), 7.00 (1H, s, HC═C) ppm.

The use of ¹⁴C methyl magnesium bromide in preparation on compound (2.1)renders the above synthesis a formal synthesis of a radioactive species.

EXAMPLE 3 Synthesis of6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-(N(2,4-dinitrophenylacyl)-3-aminopropyl)-8H-pyrimido[4,5-c][1,2]oxazin-7-one[3,6] and6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-(N-(6-(fluorescein-5-(and-6)-carboxamidohexanoyl))-3-aminopropyl)-8H-pyrimido[4,5-c][1,2]oxazin-7-one[3,7]

Preparation of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-(N-(4-methoxytriphenylmethyl)-5-amino-2(R,S)-hydroxypentyl)-2′-deoxyuridine(3.1)

To magnesium turnings (520 mgs, 217 mgram atom) in dry ether (20 ml) atroom temperature under N₂ was added a crystal of iodine followed by aslow, dropwise addition of2,2,5,5-tetramethyl-1-aza-2,5-disilacyclopentane-3-bromopropane (5.46 g,27.3 mmol) in anhydrous ether (20 ml). The Grignard preparation wasallowed to stir at room temperature under N₂ for a further 30 minutesafter addition had been completed. To the now formed2,2,5,5-tetramethyl-1-aza-2,5-disilacyclopentane-1-propylmagnesiumbromide (Ref. below) was added, by slow dropwise addition over 15minutes a solution of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyldisiloxanylidene))-5-(2-carbonylmethyl)-2′-deoxyuridinecompound (2.45 g, 4.79 mmol) and acetic acid (100 μl, 1.75 mmol) inanhydrous tetrahydrofuran (40 ml). The reaction mixture was permitted tostir for a further 30 minutes at room temperature under N₂ to ensurecompletion of the reaction. The reaction was quenched by pouring into asaturated NH₄Cl solution (250 ml) and brine (100 ml) mixture. Theorganic solvent was removed under reduced pressure and the productsubsequently extracted with dichloromethane (x3). The dichloromethaneextracts were combined, dried with magnesium sulphate and concentratedunder reduced pressure to a gum which was dried under high vacuum. Thegum was dissolved in dichloromethane (20 ml) and4-methoxytriphenylmethyl chloride (1.77 g, 5.75 mmol) and anhydroustriethylamine (3.3 ml, 23.95 mmol) were added. The reaction mixture wasleft to stir at room temperature for 18 hours. The organic solution waswashed with brine (x1), dried over magnesium sulphate and finallyconcentrated under reduced pressure to a gum. Product purification wasachieved by silica gel flash chromatography, eluant ether, to providethe title compound (3.1) as a pale yellow foam. Yield 2.45 g, 61%, Rf0.41 in ether on silica t.l.c.

δ_(H)(300 MHz; CDCl₃) 0.95-1.0(28H, m, Si—CH(CH ₃)₂), 1.4-1.6(4H, m,CH₂CH₂), 2.1-2.6(6H,m,CH₂N, ═C—CH₂, H2,H2′), 3.7-OCH ₃, H4, CH—OH),3.9-4.1(2H, m, H5), 4.5(1H, m, H3), 6.05(1H, m, H1), 6.8(2H, d, ArH),7.15-7.46(15H, m, ArH, ═CH) ppm.

Ref. F. Z. Basha and J. F. DeBernardis. Tetrahedron Lett. 25, 5271,1984.

Preparation of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-(N-(4-methoxytriphenylmethyl)-5-amino-2(R,S)-phthalimido-oxypentyl)-2′-deoxyuridine(3.2)

This was prepared in an analogous manner to3′,5′-O-(1,3-(1,1,3,3-tetraisopropydisiloxanylidene))-5-(2-(R,S)-phthalimido-oxypropyl)-2′-deoxyuridine(2.2) starting with the alcohol (3.1) (2.35 g). Purification of thecrude product by trituration in light petroleum/dichloromethane andrepetitive flash column chromatography (ethylacetate-chloroform-triethylamine 10:89:1-20:79:1 gradient) afforded thetitle compounds (3.2) (ca. 2.0 g) contaminated with triphenylphosphineoxide.

δ_(H)(300 MHz; CDCl₃) 0.81-1.13 (28H, m, 4×Me₂CH), 1.61-1.87 (4H, m,3″-CH₂ and 4″-CH₂), 2.06-2.19 (2H, m, 5″-CH₂), 2.35-2.50 (2H, m.2′-CH₂), 2.50-2.81 (2H, m, 1″-CH₂), 3.68-3.83 (1H, m, 4′-CH), 3.76 (3H,s, Ar—OMe), 3.94-4.08 (2H, m, 5′-CH₂), 4.26-4.37 (0.5H, m, 2″-CH),4.37-4.50 (0.5H, m, 2″-CH), 4.50-4.65 (1H, m, 3′-CH), 6.15 and 6.23(each 0.5H, app. t, 1′-CH), 6.71-6.82 (2H, m, ArH), 7.08-7.84 (16.5H, m,16 ArH+0.5 6-CH), 7.92 (0.5H, s, 6-CH) and 8.29 (1H, broad, NH) ppm.

Preparation of1-(3,5-O-(1,3-(1,1,3,3-tetraisopropyldisiloxanylidene))-2-deoxy-β-D-ribofuranosyl)4-triazolo-5-(N-(4-methoxytriphenylmethyl)-5-amino-2(R,S)-phthalimido-oxypentyl)-1H-pyrimidin-2-one(3.3)

This was prepared in an analogous manner to1-(3,5-O-(1,3-(1,1,3,3-tetraisopropyldisiloxanylidene))-2-deoxy-β-D-ribofuranosyl)4-triazolo-5-(2(R,S)-phthalimido-oxyhex-5-enyl)-1H-pyrimidin-2-one(2.3). Impure (3.2) (2.0 g) afforded the title compounds (3.3) (1.29 g)after flash column chromatography (ethyl acetate-light petroleum 80:20)as a sticky gum.

δ_(H)(300 MHz; CDCl₃) 0.77-1.13 (28H, m, 4×Me₂CH), 1.42-1.98 (4H, m,3″CH₂ and 4″CH₂), 2.03-2.19 (2H, m, 5″-CH₂), 2.35-2.50 (1H, m, 2′-CHH),2.50-2.68 (1H, m, 2′-CHH), 2.76 (0.5H, dd, J 15.0 and 10.3 Hz, 1″-CHH),2.98 (0.5H, dd, J 14.7 and 8.4 Hz, 1″-CHH), 3.41 (0.5H, dd, J 15.0 and3.8 Hz, 1″-CHH), 3.63-3.77 (0.5H, m, 1 ″-CHH), 3.70 (3H, s, ArOMe),3.77-3.87 (1H, m, 4′-CH), 4.92-5.23 (3H, m, 5′-CH₂ and 2″-CH), 5.38 (1H,m, 3′-CH), 6.00 (0.5H, d, J 5.5 Hz, 1′-CH), 6.13 (0.5H, dd, J 7.3 and1.8 Hz, 1′-CH), 6.73 (2H, m, ArH), 6.98-7.45 (12H, m, ArH), 7.58-7.74(4.5H, m, 0.5×6-CH and 4×phthalimideH), 7.86 (0.5H, s, 6-H), 8.18, 8.31,9.18 and 9.26 (each 0.5H, s, triazolideH) ppm.

Preparation of6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-(N-(4-methoxytriphenylmethyl)-3-aminopropyl)-8H-pyrimido[4,5-c][1,2]oxazin-7-one (3.4)

1-(3,5-O-(1,3-(1,1,3,3-Tetraisopropyldisiloxanylidene)))-2-deoxy-β-D-ribofuranosyl)4-triazole-5-(N-(4-methoxytriphenylmethyl)-5-amino-2(R,S)-phthalimido-oxypentyl)-1H-pyrimidin-2-one,(3.3) (1.29 g, 1.24 mmol) was reacted in an analogous way to example(2.4) to provide the title compounds (3.4) as a clear foam after silicagel flash chromatography using methanol dichloromethane, 5:95 as eluant.Yield 050 g. 68% Rf 0.31 in methanol:dichloromethane. 5:95 on silicat.l.c.

δ_(H)(300 MHz; CDCl₃) 1.4-1.6(4H, m, CH₂-CH₂), 2.0-2.35(6H, sugarH2,H2′, CH₂, CH₂N), 3.4(1H, m, NO—CH), 3.6-3.7(5H, m, OCH₃, sugar 2×H5),3.8(1H, m, sugar H4), 4.37(1H, m, sugar H3), 6.05(1H, m, sugar H1),6.6-6.7(3H, m, ═CH, Ar 2×H), 7.0-7.3(12H, m, ArH) ppm.

Preparation of6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-(N(2,4-dinitrophenylacyl)-3-aminopropyl)-8H-pyrimido[4,5c][1,2]oxazin-7-one(3.6).

6-(2-Deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-(N-(4-methoxytriphenylmethyl)-3-aminopropyl)-8H-pyrimido[4,5-c][1,2]oxazin-7-one(3.4) (0.26 g, 4.35 mmol) was dissolved in acetonitrile (5 ml), aceticacid (8 ml) and water (2 ml) and left to stir at room temperature for 18hours. T.l.c. (methanol: CHCl₃ 1:9) indicated that amine deprotectionhad gone to completion and that the free amino compound (3.5) remainedon the baseline. The reaction solvent was then removed under reducedpressure and the last traces of acetic acid removed by co-evaporationwith toluene (x3) followed by co-evaporation with toluene plustriethylamine (0.5 ml) (x3). The residual gum was redissolved in CH₂Cl₂(10 ml) and DMF (10 ml) and the N-hydroxysuccinimidyl ester of2,4-dinitrophenylacetic acid (300 mg, 0.93 mmol) was added and thereaction allowed to stir at room temperature for two hours. The reactionsolvent was removed by exhaustive co-evaporation with toluene. Theproduct was purified by an initial silica gel flash chromatographycolumn using CHCl₃:methanol:water, 80:18:2 followed by a final hplcpurification using a PRP-1 column and an acetonitrile:water gradient togive the title compounds (3.6) as a pale yellow gum. Yield 136 mg 59% Rf0.2 in methanol:CHCl₃ 1:9 on silica t.l.c.

δ_(H)(300 MHz;CD₃OD) 1.6-1.8(4H, m. CH₂CH₂), 2.15(2H, m, ═CH—CH ₂),2.35(1H, m, sugar H2), 2.65(1H, m, sugar H2), 3.3(2H, m, CH₂NHC(O)),3.6-3.8(3H, m, sugar 2×H5, ═N—O—CH), 3.85(1H, m, sugar H4), 407(2H, s,C(O)—CH₂—Ar). 4.35(1H, m, sugar H3), 6.25(1H, m, sugar H1), 7.09(1H, s,═CH), 7.74(1H, d, ArH, J8.4 Hz), 8.46(1H, dd, ArH, J8.4, 2.2 Hz),8.83(1H, d, ArH, J 2.2 Hz) ppm.

By standard methodology the above can be converted to a phosphoramiditeas required see P. K. T. Lin and D. M. Brown, Nucleic Acids Res. 17,10373 (1989).

Preparation of6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-(N-(6-(fluorescein-5-(and-6)-carboxamidohexanoyl))-3-aminopropyl)-8H-pyrimido[4,6-c][1,2]oxazin-7-one(3.7).

6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-(N-(4-methoxytriphenylmethyl)-3-aminopropyl)-8H-pyrimido[4,5-c][1,2]oxazin-7-one(3.4) (44 mg, 0.082 mmol) was deprotected in an analogous manner asexample (3.6), and the free amino group reacted withfluorescein-5(6)-carboxamidocaproic acid N-hydroxsuccinimide ester (65mg, 0.11 mmol). Product purification was achieved by silica gel flashchromatography using CHCl₃:methanol:water, 10:5:1 as eluant to yield thetitle compound as an orange gum. Yield 39 mg, 49%, Rf 0.49 inCHCl₃:methanol:water, 10:5:1 on silica t.l.c.

δ_(H)(300 MHz; CDCl₃) 1.3-1.7(1OH, m, —CH₂—), 2.1-2.35(5H, m, CH₂C(O),═CH—CH ₂, sugar H2), 2.55(1H, m, sugar H2′), 3.1-3.2(2H, m, CH₂NHC(O)),3.3-3.4(2H, m, CH₂NH(O)Ar), 3.5-3.×(3H, m, sugar 2×H5, ═N—O—CH), 3.8(1H,m, sugar H4), 4.35(1H, m, sugar H3), 6.25(1H, m, sugar H1), 6.5-8.4(10H,series of m, ArH, ═CH) ppm.

EXAMPLE 4 Preparation of 6-(-2-deoxy-β-D-ribofuranosyl -34-dihydro-3(R,S)-but-5-enyl-8H5pyrimido[4,5c][1,2]oxazin-7-one (4.4)

Preparation of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-(2-(R,S)-hydroxyhex-5-enyl)-2′-deoxyuridine(4.1)

Allylmagnesium chloride (25 ml of a 2.0 M solution in THF, 50 mmol) wasadded dropwise, over 15 min, to a stirred slurry of copper (I)bromide-dimethyl sulphide complex (10.3 g, 50.3 mmol) in THF (132 ml) at−78° C. After 40 min a cooled, −78° C., solution of the epoxide (1.4)(4.41 g, 8.38 mmol) in THF (80 ml) was added via a cannula over 15 min,followed by a THF wash (10 ml). The reaction mixture was allowed to stirat −78° C. for a further 70 min after which the cooling bath was removedand the reaction mixture was allowed to warm to ambient over a period of60 min. The mixture was then added via a cannula to a vigorously stirredmixture of Et₂O (250 ml), saturated NH₄Cl (aq) (230 ml) and conc. NH₄OH(20 ml). The reaction vessel was washed with a mixture of Et₂O (20 ml),saturated NH₄Cl (aq) (20 ml) and conc. NH₄OH (20 ml) which was added tothe rest of the material. The mixture was then stirred for a further 30min and diluted with Et₂O. The aqueous phase was separated, exhaustivelyextracted with Et₂O and the combined organic phase was washed withbrine, dried (MgSO₄) and concentrated in vacuo. Both TLC and ¹H nmrevidence suggested that the required product was contaminated with asubstantial amount of the bromohydrin. Brief treatment of the crudematerial with methanolic K₂CO₃, followed by a standard extractive workup, afforded a crude mixture containing the starting epoxide (1.4), therequired title compounds (4.1) and other material thought to be due topartial cleavage of the siloxy moiety. Flash column chromatography ofthe crude product (dichloromethane-methanol 100:0-95:5 gradient)afforded the title compound contaminated with the starting epoxide (2.72g total).

δ_(H)(300 MHz; CDCl₃) 0.82-1.16 (28H,m,4×Me₂CH), 2.00-2.58 (8H, m,2′-CH₂, 1″+3″+4″-CH₂), 3.68-3.82 (1H, m, 4′-CH), 3.93-4.13 (2H, m,5′-CH₂), 4.42-4.58 (1H, m, 3′-CH₂), 4.90-5.10 (2H, m, 6″-CH₂), 5.73-5.90(1H, m, 5″-CH), 7.39 and 7.44 (each 0.5H, s, 6-CH) and 8.18 (1H, br s,NH) ppm.

Preparation of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-(2-(R,S)-phthalimido-oxyhex-5-enyl)-2′-deoxyuridine(4.2)

This compound was prepared in an analogous manner to 3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-(2-(R,S)-phthalimido-oxypropyl)-2′-deoxyuridine(2.2) using3′,5′-O-(1,3-(1,1,3,3,-tetraisopropyidisiloxanylidene))-5-(2-(R,S)-hydroxyhex-5-enyl)-2′-deoxyuridine(4.1) contaminated with the epoxide (1.4) (2.70 g total). Repetitiveflash column chromatography (ethyl acetate-light petroleum 1:5-7:3gradient and dichloromethane-methanol 100:0-98.5:1.5 gradient) followedby trituration in hexane/dichloromethane, filtration and concentrationof the filtrate afforded the title compounds (4.2) (3.32 g) as a whitesolid, which was still contaminated with the epoxide (1.4).

δ_(H)(300 MHz; CDCl₃) 0.77-1.16 (28H, m, 4×Me₂CH), 2.18-2.68 (8H, m,2′-CH₂, 1″+3″+4″-CH₂), 3.68-3.85 (1H, m, 4′-CH), 3.95-4.11 (2H, m,5′-CH₂), 4.35-4.63 (1H, m, 3′-CH), 4.90-5.13 (2H, m, 6″-CH₂), 5.81 (1H,m, 5″-CH), 6.13 and 6.23 (each 0.5H, m, 1′-CH), 7.39 and 7.44 (each0.5H, s, 6-CH), 7.71-7.84 (4H, m, ArH) and 8.16-8.27 (1H, broad, NH)ppm.

Preparation of1-(3,5-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-2-deoxy-β-D-ribofuranosyl)-4-triazolo-5-(2(R,S)-phthalimido-oxyhex-5-enyl)-1H-pyrimidin-2-one(4.3)

This was prepared in an analogous manner to1-(3,5-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-2-deoxy-β-D-nbofuranosyl)4-triazolo-5-(2(R,S)-phthalimido-oxyhex-5-enyl)-H-pyrimidin-2-one(2.3) using the impure 2′-deoxyuridine (4.2) (3.3 g). Reaction progresswas monitored by TLC ( ethyl acetate) and upon completion the mixturewas filtered and the solids washed with Et₂O The filtrate wasconcentrated in vacuo and the residue was subjected to a standardextractive work up (Et₂O/H₂O). Flash chromatography (ethyl acetate-lightpetroleum 1:1-7:3 gradient) of the crude product afforded the titlecompounds (4.3) (1.33 g pure and 1.1 g of impure material) as stickysolids.

δ_(H)(300 MHz; CDCl₃) 0.81-2.16 (28H, m, 4×Me₂CH), 1.69-2.05 (2H, m,3″-CH₂), 2.05-2.55 (3H, m, 4″-CH₂+2′CHH), 2.55-2.71 (1H, m, 2′-CHH),2.85 (0.5H, dd, J 15.0 and 9.9 Hz, 1″-CHH), 3.05 (0.5H, dd, J 14.7 and8.4 Hz, 1″-CHH), 3.47(0.5H, dd, J 14.8 and 4.1 Hz, 1″-CHH, 3.71 (0.5H,dd, J 14.9 and 2.6 Hz, 1″-CHH), 3.79-3.90 (1H, m, 4′-CH), 3.94-4.24 (3H,m, 5′-CH₂₊₂′-CH), 4.39 (1H, m, 3′-CH), 4.90-5.10 (2H, m, 6″-CH₂), 5.79(1H, m, 5″-CH), 6.04 (0.5H, dd, J6.7 and 1.5 Hz, 1′-CH), 6.17 (0.5H, dd,J 7.4 and 2.10 Hz, 1′-CH), 7.65-7.81 (4.5H, m, 4×ArH+0.5 6-CH), 7.95(0.5H, s, 6-CH), 8.21, 8.33, 9.23 and 9.30 (each 0.5H, s, triazolide H)ppm.

Preparation of6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-but-5-enyl-8H-pyrimido[4,5c][1,2]oxazin-7-one(4.4)

This was prepared in an analgous manner to6-(2-deoxy-β-β-ribofuranosyl)-3,4-dihydro-3(R,S)-methyl-8H-pyrimido[4,5c][1,2]oxazin-7-one(2.4). Triazolide (4.3) (1.30 g) afforded, after flash columnchromatography (dichloromethane-methanol 99:1-95:5 gradient) theprotected cyclised material as a sticky foam (930 mg).

Partial proton NMR data: δ_(H)(300 MHz; CDCl₃) 0.85-1.20 (28H, m,4×Me₂CH), 1.53-1.69 (1H,m, 1″-CHH), 1.75-1.90 (1H, m, 1″-CHH), 2.12-2.61(6H, m, 3′-CH₂+4-CH₂+2″-CH₂), 4.03 (2H, m, 5′-CH₂), 4.46 (1H, m, 3′CH),4.95-5.14 (2H, m, 6″-CH₂), 5.83 (1H, m, 5″-CH), 6.08 (1H, m, 1′-CH),6.73 and 6.76 (each 0.5H, s, 6-CH) and 8.53 (1H, br s, NH)

Treatment of a portion of this material (ca. 450 mg) withtetra-n-butylammonium fluoride furnished the title compounds (4.4) (200mg), as a sticky gum, after flash column chromatography(dichloromethane-methanol 95:5-90:10 gradient).

UV λ_(max) (MeOH) 204, 232 and 298 nm

δ_(H)(300 MHz;CD₃OD) 1.53-1.79 (2H, m, 1″-CH₂), 2.10-2.45 (5H, m,4-CHH+2′-CH₂+2″-CH₂), 2.65 (1H, br d, J 14.4 Hz, 4-CHH), 3.5-3.82 (3H,m, 3-CH+5′-CH2) 3.87 (1H, m, 4′-CH), 4.35 (1H, m, 3′-CH), 4.96 (1H, brd, J 10.3 Hz, 4″-CHH), 5.04 (1H, dd, J 16.9 and 1.7 Hz, 4″-CHH), 5.74(1H, ddt, J 16.9,10.3 and 6.6 Hz, 3″-CH), 6.26 (1H, m, 3′-CH), 7.1 (1H,s, 4-H) ppm.

δ_(c)(75 MHz; CD₃OD) 30.24, 30.38, 30.50, 33.96, 34.01, 40.53, 62.99,72.32, 72.34, 75.95, 85.53, 88.36,101.70,101.76, 115.52, 130.46, 139.07,151.44, 151.51, 152.63 and 152.65 ppm.

EXAMPLE 5 Synthesis of6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-(3,3-diO-(1,3-propylidene)-propyl)-8H-pyrimido[4,5c][1,2]oxazin-7-one

Preparation of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-5-(5,5-diO-(1,3-propylidene)-2(R,S)-hydroxypentyl)-2′-deoxyuridine(5.1)

Into an oven-dried 3-neck 50 ml flask were added magnesium turnings(0.60 g, 25 mgram atom) and dry diethyl ether (5 ml ), and the mixtureset stirring under an argon atmosphere. To this was added a solution offreshly distilled 2-(2-bromoethyl)-1,3-dioxane (4.88 g, 25 mmol) in drytetrahydrofuran (15 ml), in portions so as to control the exothermicreaction that ensued. Once all the bromide had been added the reactionwas allowed to subside before being heated at reflux for 30 minutes (hotwater bath). By the end of this time almost all of the magnesium hadbeen consumed to give a pale yellow solution.

About half of this solution (12 mmol Grignard reagent) was transferredto a second dry, argon-filled flask. To this was added a solution of3′,5′-O-(1,3-(1,1,3,3-tetraisopropyldisiloxanylidene))-5-(carbonylmethyl)-2′-deoxyuridine(1.3) (1.64 g, 3.2 mmol) in dry tetrahydrofuran (5 ml) and acetic acid(40 μl). The resulting mixture was stirred for 30 minutes, then quenchedwith saturated aqueous ammonium chloride solution and extracted withdiethyl ether. The combined ether extracts were dried (Na₂SO₄), filteredand evaporated under reduced pressure to a yellow oil. Purification byflash column chromatography (silica; 25% light petroleum/ethyl acetate)gave the title compound (5.1) as an off-white foam, 1.37 g (68%). UV(CHCl₃) 270 nm.

δ_(H)(300 MHz; CDCl₃) 0.8-1.1 (28H, m, Si—iPr×4), 1.31 (1H, m,propylidene O-CH₂-CHH-CH₂-O), 1.40-1.75 (5H, m), 2.06 (1H, m,propylidene O-CH₂-CHH-CH₂-O), 2.23-2.58 (4H, m), 3.28 (1H, broad t, OH),3.70-3.78 (4H, m, sugar 4′+-CHOH +propylidene O-CH ₂-CHH-CH₂-O),4.00-4.10 (4H, m, sugar 5′+propylidene O-CH₂-CHH-CH ₂-O), 4.48 (1H, m,sugar 3′), 4.56 (1H, t, J4.5 Hz, acetal O-CH-O), 6.05 (1H, m, sugar 1′),7.36+7.42 (1H, 2s, C6 diastereomers), 8.60+8.62 (1H, 2s, N3diastereomers) ppm.

Preparation of3′,5′-O-(1,3-(1,1,3,³-tetraisopropyidisiloxanylidene))-5-(5,5-diO-(1,3-propylidene)-2(R,S)-phthalimido-oxypentyl)-2′-deoxyuridine(5.2)

Prepared as for3′,5′-O-(1,3-(1,1,3,3-tetraisopropyldisiloxanylidene))-5-(2-(R,S)-phthalimido-oxypropyl)-2′-deoxyuridine(2.2). UV (CHCl₃) 242, 268 nm.

δ_(H)(300 MHz; CDCl₃) 0.86-1.11 (28H, m, Si—iPr×4), 1.23-1.30 (1H, m,propylidene O-CH₂-CHH-CH₂-O), 1.70-2.10 (5H, m), 2.37-2.71 (4H, m),3.66-3.81 (3H, m, sugar 4′+propylidene O-CH ₂-CHH—CH ₂O), 3.96-4.08 (4H,m, sugar 5′+propylidene O—CH₂-CHH-CH ₂-O), 4.33-4.50 (1H, 2m,-CH(O-phthalimide) diastereomers), 4.50-4.60 (2H, m, sugar 3′+acetalO—CH-O), 6.13+6.22 (1H, 2 apparent t, sugar 1′ diastereomers), 7.70-7.86(5H, m, C6+phthalimide 4H), 8.44+8.46 (1H, 2s, N3 diastereomers).

Preparation of1-(3′,5′-O-(1,3-(1,1,3,3-tetraisopropyidisiloxanylidene))-2-deoxy-β-D-ribofuranosyl)4-triazolo-5-(5,5-diO-(1,3-propylidene)-2(R,S)-phthalimido-oxypentyl)-1H-pyrimidin-2-one(5.3)

Prepared as for1-(3′,5′-O-(1,3-(1,1,3,3-tetraisopropyldisiloxanylidene))-2-deoxy-β-D-ribofuranosyl)-4-triazolo-5-(2-(R,S)-phthalimido-oxypropyl)-1H-pyrimidin-2-one(2.3). Used directly in the next step.

Preparation of6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-(3,3-diO-(1,3-propylidene)-propyl)-8H-pyrimido[4,5c][1,2]oxazin-7-one(5.4)

Prepared as for6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-methyl-8H-pyrimido[4,5c][1,2]oxazin-7-one(2.4). Triazolide (5.3) (0.50 g) afforded, after treatment with1,4-dioxan saturated with ammonia, and purification by flash columnchromatography (silica; 40% ethyl acetate/dichioromethane), theprotected cyclised product (0.28 g). UV (CHCl₃) 302 nm. δ_(H)(300 MHz;CDCl₃) 0.89-1.07 (28H, m, Si—iPr×4), 1.31 (1H, m), 1.50-2.53 (9H, m),3.65-3.78 (4H, m, sugar 4′+ring 3-H+propylidene O-CH ₂-CHH—CH₂-O),3.94-4.10 (4H, m, sugar 5′+propylidene O—CH₂-CHH-CH ₂-O), 4.43 (1H, m,sugar 3′), 4.55 (1H, broad t, acetal O—CH-O), 6.03 (1H, m, sugar 1′),6.66+6.69 (1H, 2s, C5 diastereomers), 7.5-8.0 (1H, broad s) ppm.

Treatment of this material with tetra-n-butylammonium fluoride gave thetitle compounds (100 mg). Complete removal of the tetra-n-butylammoniumions was not possible.

EXAMPLE 6 Synthesis of6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4.5-c][1,2]pyridazin-7-one(6.4) and5-(2-chloroethyl)-1-(2′,3′-isopropylidine-β-D-ribofuranosyl)uridine(6.8)

Preparation of5-(2-chloroethyl)-1-(3′,5′-diO-dimethoxytrityl-2′-deoxy-β-D-ribofuranosyl)uridine(6.1)

5-(2-Chloroethyl)-2′deoxyuridine (2 g, 6.9 mmol) was dissolved inpyridine (40 ml) and dimethoxytrityl chloride (79, 21 mmol) added andthe solution heated at 50° C. overnight. The solvent was evaporated andthe product dissolved in chloroform and washed with sodium bicarbonatesolution, dried and evaporated under reduced pressure to a yellow gumwhich was chromatographed (CHCl3) to give a yellow foam. Yield 5.8 g,94%.

Preparation of1-(3′,5′-diO-dimethoxytrityl-2′-deoxy-β-D-ribofuranosyl)-4-(1,2,4-triazolo)-5-(2-chloroethyl)-pyramid-2-one(6.2).

To an ice-cold suspension of 1,2,4-triazole (6.4 g, 9.3 mmol) inacetonitrile (100 ml) was added phosphorous oxychloride (1.7 ml, 2.8mmol) and the solution stirred at 0° C. for 15 minutes. To this was thenadded triethylamine (1 5.5 ml, 11.2 mmol) and the solution stirred for afurther 15 minutes. The tritylated nucleoside (6.1) (5.5 g, 6.1 mmol) inacetonitrile (25 ml) was added and the solution stirred at roomtemperature overnight. The solvent was removed and the product dissolvedin chloroform and washed (sodium bicarbonate), dried (sodium sulphate)and evaporated to a brown gum which was chromatographed (CHCl3) to givea yellow foam. Yield 6.1 g, 105%.

Preparation of1-(2-deoxy-β-D-ribofuranosyl)4-(1,2,4-triazolo)-5-(2-chloroethyl)-pyramid-2-one(6.3).

To a solution of the above nucleoside (6.2) (6 g, 6.3 mmol) indichloromethane (100 ml) was added trichloroacetic acid (5.2 g, 32 mmol)and the solution stirred at room temperature overnight. The solution wasconcentrated and chromatographed (CHCl₃ then CHCl₃/5% MeOH) to give awhite solid. Yield 1.73 g, 80%.

δ_(H)(DMSO-d6) 2.09-2.17, 2.35-2.41 (2H, m, H2′, H2″), 3.20-3.30 (2H, m,C5-CH₂), 3.58-3.77 (4H, m, H5′, H5″, CH₂Cl), 3.89-3.93 (1H, m, H3′),4.23-4.28 (1H, m, H4′), 5.23 (1H, br. s, OH), 5.32 (1H, br. s, OH), 6.11(1H, t, J 5.9 Hz, H1′), 8.40 (1H, s, triazolo CH), 8.69 (1H, s, H6),9.33 (1H, s, triazolo CH) ppm. UV λ_(max) 321 (ε=5600), 249 (ε=6500),ε260 (μM)=4.9. pH 12 (irreversible) λ_(max) 267.

A small amount of the triazolo nucleoside (6.3) was dissolved inacetonitrile and treated with anhydrous hydrazine to give the bicyclicproduct (6.4). UV λ_(max) 280 (ε=14800).

Preparation of5-(2-chloroethyl)-1-(2′,3′,5′-tri-O-acetyl-β-D-ribofuranosyl)uridine(6.6)

5-(2-Chloroethyl)uracil (6.5) (0.5 g, 2.9 mmol), formed by the method ofGriengl (J Med Chem vol 28, pp1679-1684, 1985), was heated at 120° C.overnight with hexamethyldisilazane (20 ml) and chlorotrimethisilane(1.5 ml). This was cooled to room temperature and evaporated, thencoevaporated with dry xylene( 3×25 ml) and then dissolved in dryacetonitrile (25 ml). 1,2,3,5-tetra-O-acetyl ribose (1.0 g, 3.1 mmol)was dissolved in dry acetonitrile (25 ml) and then sodium iodide (1.29g, 8.6 mmol) added and allowed to dissolve. To this was then addedchlorotrimethylsilane (0.55 ml, 4.3 mmol) and the solution stirred atroom temperature for 20 minutes. This was then added to the silylatedbase and the whole stirred at room temperature for 2 hours. TLC showsone major spot. The solution was then evaporated redissolved inchloroform and extracted firstly with sodium bicarbonate solution andthen with sodium thiosulphate solution, dried and evaporated to dryness.The residue was lo then purified by column chromatography (silica,chloroform to chloroform/2% methanol gradient) to isolate the product asa white foam. Products were observed on t.l.c. by staining withp-anisaldehyde solution (anisaldehyde, sulphuric acid, ethanol;1:1:10)and heating the plate. Yield 0.88 g, 71%.

δ_(H)(DMSO-d6) 2.00, 2.04, 2.07(9H, 3×s, 3×COCH₃), 2.66(2H, t, J6.9 Hz,5-CH₂), 3.70(2H,t, J6.9Hz, CH₂Cl), 4.18-4.33(3H, m), 5.31-5.36(1H, m),5.41-5.45(1H,m), 5.89(1H, d, J 5.2 Hz, 1′-CH), 7.66(1H, s, 6-CH),11.56(1H, s, NH) ppm.

Preparation of 5-(2-chloroethyl)-1-(β-D-ribofuranosyl)uridine (6.7)5-(2-chloroethyl)-1-(2′, 3′,5′-tri-O-acetyl-β-D-ribofuranosyl)uridine(15 g, 34.76 mmol) was dissolved in a solution of potassium carbonate(0.5M in methanol/water, 3:1) (350 ml). After 2 hours, t.l.c.(dichloromethane/methanol, 9:1) showed that all starting material hadbeen converted to a product near the baseline. Pre-washed Dowex 50 W X8ion exchange resin (H+form) was added to neutralise the base as verifiedby the pH of the solution. The solid resin was filtered off and washedwith a portion of methanol/water. 3:1. Methanol was evaporated off invacuo and the solution diluted with water (300 ml). The aqueous solutionwas than extracted with dichloromethane (2×100 ml) and the aqueous layerevaporated to dryness. The solid residue was then recrystallized fromethanol, yielding colourless title compound (6.7) which was homogeneousby hplc (mp158-160° C.). Yield 7.7 g, 76%. Preparation of5-(2-chloroethyl)-1-(2′,3′-isopropylidine-β-D-ribofuranosyl)uridine(6.8)

5-(2-chloroethyl)-1-(β-D-ribofuranosyl)uridine (6.7) (0.85 g, 2.8 mmol)was dissolved in acetone (30 ml) and p-toluene sulphonic acid hydrate(0.506 g, 2.66 mmol) was added, followed by triethyl orthoformate (1.66g, 11.2 mmol). The initially insoluble nucleoside dissolved within 15minutes to give a slightly yellow solution. After stirring for 2.5 hourst.l.c. (dichloromethane/methanol; 9:1) showed that all starting materialhad been converted to a new spot Rf 0.52 The volatile material wasevaporated off in vacuo and the residue treated with dipotassiumhydrogen phosphate solution (25 ml of aqueous solution containing 0.815g phosphate) and extracted with ethyl acetate (2×30 ml). The organicextract was washed with water (2×40 ml) followed by brine (40 ml) andthe organic layer dried over sodium sulphate. After filtering andevaporation of the extract to dryness, the product was purified bychromatography over silica gel eluting with dichloromethane/acetonitrile7:3 to yield title compound as a colourless solid (0.8 g, 86%). T.l.c.(dichloromethanel acetonitrile; 7:3, Rf 0.21); purity by h.p.l.c. (C-18column, 40% acetonitrile, 60% 0.1M triethylammonium acetate)>98%.

δ_(H)(CDCl₃) 7.54(1H, s), 5.75(1H, d), 4.81(2H, m), 4.18(1H, m),3.80-3.55(4H, m), 2.66(2H, m), 1.49(3H, s), 1.26(3H, s) ppm. δ_(13C)(CDCl₃) 163.488 (C), 150.367(C), 139.650(CH), 113.759(C), 110.271(C),92.957(CH), 86.480(CH), 84.108(CH), 80.352(CH), 61.952(CH₂),42.683(CH₂), 30.352(CH₂), 27.069(CH₃) 25.139(CH₃).

EXAMPLE 7 Synthesis of2-benzyl-6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]1pyridazin-7-oneand2-(3-hydroxybenzyl)-6-(3.5-di-O-p-toluoyl-2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2pyridazin-7-one

Preparation of1-(3,5-di-O-p-toluoyl-2-deoxy-β-D-ribofuranosyl)4-(1,2,4-triazolo)-5-(2-chloroethyl)-pyrimid-2-one(7.1).

1,2,4-Triazole (12.1 g, 176 mmol) was suspended in acetonitrile (250 ml)at 0° C. and to this was added phosphorous oxychloride (3.8 ml, 40 mmol)and the solution stirred at 0° C. for 15 minutes. To this was then addedtriethylamine (30 ml, 214 mmol) and the solution stirred at 0° C. for afurther 30 minutes. To this was then added a solution of3′,5′-di-O-p-toluoyl-2′-deoxyribofuranosyl-5-(2-chloroethyl)-uracil (5.3g, 10.5 mmol) in acetonitrile (50 ml) and the solution stirred at roomtemperature overnight. The solution was evaporated, dissolved inchloroform and washed with aqueous sodium bicarbonate, dried andevaporated to a gum, which was purified by chromatography (CHCl₃/2%MeOH) to give a white foam. Yield 5.82 g, 96%.

δ_(H) 1.7 (1H, s, ), 2.35 (3H, s, ), 2.42 (3H, s, ), 3.16 (1H, s,), 3.20(2H, ), 3.65 (2H, ), 4.68 (1H, d, ), 4.70 (1H, ), 4.88 (1H, ), 5.60(1H,), 6.35 (1H, ), 7.18 (2H, ), 7.25 (2H), 7.82 (2H, ), 7.95 (2H, ),8.15 (1H,), 8.20 (1H, ), 9.3 (1H, ) ppm. FAB mass 578.4, C₂₉H₂₈CIN₅O₆requires 577.5, Accurate mass measurement 577.17279.

Preparation of2-benzyl-6-(3,5-di-O-p-toluoyl-2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]pyridazin-7-one(7.2).

To a solution of the above triazole (7.1) (6.35 g, 11 mmol) andbenzylhydrazine dihydrochloride (2.35 g, 12.1 mmol) in ethanol (200 ml)was added triethylamine (6 ml) and the solution heated at refluxovernight. The solution was evaporated and chromatographed (EtOAc:hexane1:1 then CHCl₃/MeOH 2-5%) to give 4 products. Product 1 was identifiedas the C⁴-O-ethyl derivative (0.76 g), compound 3 as the 5-memberedcyclised product (2.9 g), compound 4 is un-identified (3.1 g). Compound2 was identified as the desired product (1.29 g. 20%); δ_(H) (DMSO-d6)2.23-2.56 (6H, m, CH₂N, C5-CH₂, H2′, H2″), 2.35 (3H, s, CH₃), 2.37 (3H,S, CH₃), 3.92 (2H, s, CH₂Ph), 4.36-4.38 (1H, m, H4′), 4.46-4.61 (2H, m,H5′, H5″), 5.52-5.54 (1H, m, H3′), 6.26 (1H, t, J6.5 Hz, H1′), 6.58 (1H,s, H6), 7.21-7.35 (9H, m, PhthCH, ArCH), 7.86-7.91 (4H, m, ArCH), 10.13(1H, s, NH) ppm. FAB mass 595.2 (M+H).

Preparation of2-benzyl-6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]pyridazin-7-one(7.3).

The above compound (7.2) (1.4 g, 2.35 mmol) was suspended in methanol(50 ml) and to this was added sodium methoxide (140 mg, 2.6 mmol) andthe solution stirred at room temperature for 1 hour. The solvent wasremoved and the product chromatographed (CHCl₃/10% MeOH) to give a paleyellow powder. Yield 0.61 g, 72%. δ_(H) 1.88-2.03 (2H, m, H2′, H2″),2.45-2.50 (2H, m, CH₂N), 2.59-2.63 (2H, m, CH₂C(5)), 3.41-3.54 (2H, m,H5′, H5″), 3.65-3.66 (1H, m, H4′), 3.92 (2H, s, CH₂Ph), 4.16 (1H, m,H3′), 4.89 (1H, t, 5′-OH), 5.15 (1H, d, 3′-OH), 6.13 (1H, t, J6.1 Hz,H1′), 6.74 (1H, s, H6), 7.21-7.34 (5H, m, Ph), 9.98 (1H, s, NH) ppm. UVλ_(max) 295 (ε=7800). ε260 (μM)=5.7

Preparation of2-(3-hydroxybenzyl)-6-(3,5-di-O-p-toluoyl-2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]pyridazin-7-one(7.4).

To a solution of the triazole (7.1) (1 g, 1.7 mmol) and3-hydroxybenzylhydrazine dihydrochloride (0.73 g, 3.5 mmol) in ethanol(25 ml) was added triethylamine (0.72 ml, 5.2 mmol) and the solutionheated at 50° C. for two days. The solution was evaporated under reducedpressure and purified by chromatography (dichloromethane:ethyl acetate1:1 then CHCl₃/MeOH 2%) to give 2 products. Product 1 was identified asthe 5-membered cyclised product (0.57 g, 54%). Compound 2 was identifiedas the desired product (7.4), (0.39 g, 37%). δ_(H)(DMSO-d6) 2.36, 2.38(6H, 2×s, 2×ArCH3), 2.43-57 (4H, m, H2′, H2″, C5-CH₂), 3.45 (2H, t, J7.6 Hz, N—CH₂), 3.93 (2H, d, J4.7 Hz, CH₂Ph), 4.43-4.63 (3H, m, H4′,H5′, H5″), 5.54-5.57 (1H, m, H3′), 5.67 (1H, t, J4.7 Hz, NH), 6.36 (1H,t, J6.7 Hz, is H1′), 6.63 (1H, d, J7.7 Hz, Ph-H6), 6.75-6.78 (2H, m,Ph-H2, Ph-H4), 7.09 (1H, t, J 7.7 Hz, Ph-H5), 7.30-7.37 (5H, m, 4×ArCH,H6), 7.85-7.92 (4H, m, ArCH), 9.34 (1H, s, OH) ppm.

EXAMPLE 8 Synthesis of 1 and2-(6′-(fluorescein-5-carboxamidohexanoyl))-6-(2′-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]pyridazin-7-one-5′-triphosphate(8.5)

Preparation of6-(3,5-diacetyl-2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]pyridazin-7-one(8.1)

1-(3,5-Di-O-acetyl-2-deoxyribofuranosyl)4-(1,2,4-triazolo)-5-(2-chloroethyl)pyrimido-2-one(P. Kong Thoo Lin and D. M. Brown, Nucleic Acids Res. 17, 10373-10383,1989) (0.25 g, 0.6 mmol) was dissolved in dry dichioromethane (10 ml)and anhydrous hydrazine (37 μl, 1.2 mmol) added and the solution stirredat room temperature for 30 minutes. The solution was concentrated andpurified by chromatography (chloroform/1 0% methanol) to give the titlecompound (8.1) as a white solid. Yield 0.2 g, 100%.

δ_(H) (DMSO-d6) 1.96, 2.01 (6H, 2×s, 2×CH₃CO), 2.15-2.45(2H, m, H2′,H2″), 2.75(2H, t, J 6.8 Hz, CH₂), 3.6(1H, br, s, NH), 3.83(2H, t, J6.8Hz, CH₂N), 4.12-4.14(1H, m, H4′), 4.214.24(2H, m, H5′, H5″),5.17-5.20(1H, m, H3′), 6.18(1H, t, J6.2 Hz, H1′), 7.17(1H, s, H6),9.72(1H, s, NH) ppm. m/z 352 M+. Accurate mass measurement found352.1376 (C₁₅H₂0N₄O₆) deviation −0.7 ppm. UV λmax 279.6 (ε=14800).

Preparation of 1 and2-(6-(trifluoroacetamido)hexanoyl)-6-(3′,5′-diacetyl-2′-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]pyridazin-7-one(8.2)

To a stirred solution of the 3′,5′-diacetyl nucleoside (8.1) (350 mg, 1mmol) in anhydrous dichloromethane (25 ml) was added theN-hydroxysuccinimidyl ester of N-(trifluoroacetyl)-6-aminohexanoic acid(356 mg, 1.1 mmol) and triethylamine (110 mg, 1.1 mmol) and the mixturewas stirred at room temperature for 4 hours. The solution was thenconcentrated to dryness and thin-layer chromatography of the reactionmixture in 5% methanol/chloroform indicated the presence of two minorand one major product. The major product was purified by columnchromatography to give a white solid compound though the positionalisomer cannot be specified from the data available.

δ_(H)(CDCl₃) 1.27-1.79 (6H, m, 3×CH₂), 2.10, 2.14 (6H, 2×s, 2×COCH₃),2.12, 2.41 (2H, m, H2′, H2″), 2.73 (2H, t, J6.8 Hz, NCOCH₂), 3.22-3.47(3H, m, CH₂N), 3.70 (2H, t, J6.8 Hz, C5-CH₂), 4.14-4.43 (3H, H5′,H5″,H3′), 5.11-5.25(1H,m,H4′),6.24 (1H, t, J6.6 Hz, H1′), 7.31 (1H, s, H6)9.56, 11.38 (2×s, NH) ppm.

Preparation of 1 and2-(6-(trifluoroacetamido)hexanoyl)-6-(2′-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]pyridazin-7-one(8.3)

The 3′,5′-diacetyl nucleoside containing the N-trifluoroacetyihexanoyllinker (8.2) (500 mg, 0.9 mmol) was dissolved in methanol (20 ml) and tothe stirring solution, at room temperature, was added a methanolicsolution of sodium methoxide (2.0 ml, 0.5M, 1.0 mmol). After 20 minutes,the solution was evaporated under reduced pressure and the productchromatographed on a silica gel column (CHCl₃/10% methanol) to give thetitle compound (8.3) (250 mg, 56%) as a white solid.

δ_(H) (DMSO-d6)1.27-1.52 (6H, m, 3×CH₂), 1.84-2.50 (2H, m, H2′, H2″),2.18 (2H, t, J6.8 Hz, NCOCH₂), 2.85 (2H, m, CH₂N), 3.19 (1H, m, CHN),3,39 (1H, m, CHN), 3.52 (2H, m, C5-CH₂), 3.70-3.81 (3H, m, H5′,H5″,H3′),3.394.01 (1H, m, H4′), 6.18 ( 1H, t, J 6.6 Hz, H1′), 7.71 (1H,s, H6), 9.43,10.22 (2×s NH) ppm.

Preparation of 1 and2-(6-aminohexanoyl)-6-(2′-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]pyridazin-7-one-5′-triphosphate( 8.4)

To a stirred and cooled (0° C.) solution of2-(6-(trifluoroacetamido)hexanoyl)-6-(2′-deoxy-β-D-furanosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]pyridazin-7-one(8.3) (220 mg, 0.46 mmol) in trimethylphosphate (2 ml) was added POCl₃(65 μl. 0.69 mmol). After one hour, the reaction mixture wassimultaneously treated with 0.5M DMF solution of bis-n-tributylammoniumpyrophosphate (4.72 ml, 2.30 mmol) and n-tributylamine (547 ml, 2.30mmol). After stirring the reaction mixture at room temperature for 10minutes, it was neutralised with 1.0M TEAB (triethylammoniumbicarbonate) and stirred at room temperature overnight, evaporated underreduced pressure and the residue obtained was dissolved in water (30ml). Thus, the crude triphosphate was loaded on a Sephadex column (500ml) and the desired triphosphate eluted using the gradient 0.05 (2L) to1.0M TEAB (2L, pH=7) at 2 ml/min flow rate. After characterising it by³¹P nmr, δ_(p) (D₂O/EDTA) −10.36 (d, γ-P), −10.86 (d, (α-P), −22.79 (t,β-P) ppm, the triphosphate was treated with 30% NH₄OH (6 ml) overnightfor the deprotection of the amino group. The reaction mixture wasevaporated under reduced pressure and the residue obtained was purifiedby semi-prep-HPLC using Waters Delta Pak 15 micron C18 column (5cm×30cm)under the gradient conditions of 0-00% buffer A (0.1 M TEAB) and bufferB (25% CH₃CN in 0.1M TEAB) at 130 ml/min over 30 minutes. The desiredtriphosphate (8.4) fractions were pooled, evaporated and lyophilised toget pure (8.4) (83 mg, 19.45%) as the triethylammonium salt.

Preparation of 1 and 2-(6′-(fluorescein-5-carboxamidohexanoyl))-6-(2′-deoxy-β-D-ribofuranosyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]pyridazin-7-one-5′-triphosphate(8.5)

To a stirred solution of the triphosphate (8.4) (5 mg, 0.05 mmol) in0.2M Na₂CO₃-NaHCO₃ buffer (800 μpH 8.5) was added an anhydrous DMFsolution (600 μl) of the N-hydroxysuccinimidyl ester of5-carboxyfluorescein (10 mg, 0.02 mmol) at room temperature and stirringcontinued overnight. After evaporating the reaction mixture underreduced pressure, the yellow coloured residue obtained was dissolved ina minimum amount of 1:1 aqueous methanol, loaded on a glass column (40cm×2 cm) packed up to 20 cm height with 35-70 microns silica gel 60(EM-Separations, cat no. 9389-5). The excess dye was eluted using 1:1chloroform/methanol to neat methanol and the desirednucleotide-fluorescein conjugate was eluted using 6:3:1 i-PrOH:NH₄OH:H₂Oto obtain (8.4) as a yellow solid after pooling and evaporation.Compound (8.5) was further purified by HPLC on a 15 microns Delta PakC18 column (1.9 cm×30 cm) under the gradient conditions of 0-50% bufferA (0.1 M TEAB, pH 7.1) and buffer B (25% acetonitrile in 0.1M TEAB, pH7.0) at 15 ml per minute in 30 minutes. The desired compound (8.5)fractions were pooled, evaporated and lyophilised to get pure (8.5) as ayellow solid (quantitative yield).

EXAMPLE 9 Synthesis of Nucleoside Triphosphates

All the following triphosphates were prepared in an analogous manner tothe triphosphate example (8.4)

6-(2-Deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-methyl-8H-pyrimido[4,5-c][1,2]oxazin-7-one-5′-triphosphate(9.1)

Derived from nucleoside (2.4)

δ_(p)(121 MHz;D₂O) −10.91 (d, γ-P), −11.51 (d, α-P) and −23.33 (t, β-P)

6-(2-Deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-but-5-enyl-8H-pyrimido[4,5c][1,2]oxazin-7-one-5′-triphosphate(9.2)

Derived from nucleoside (4.4)

δ_(H)(300MHz; D₂O/KOH) 1.65 (2H, m, allyl), 2.0-2.8 (6H, m), 3.65 (1H,m, sugar). 4.0 (3H, m, sugar+H3), 4.5 (1H, broad, sugar). 4.9 (2H, dd,allyl), 5.75 (1H, m, allyl), 6.2 (1H, m, sugar), 7.0 (1H, s) ppm.

δ_(p)(121 MHz:D₂O/KOH) −5.84 (d, γ-P), −11.00 (d, α-P) and −21.88 (t,β-P)

6-(2-Deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-(N(2,4-dinitrophenyacyl)-3-aminopropyl)-8H-pyrimido[4,5-c][1,2]oxazin-7-one-5′-triphosphate(9.3)

Derived from nucleoside (3.6)

δ_(H)(300 MHz; D₂O) 2.0-2.4 (3H, m), 2.6-3.0 (4H, m), 3.8 (1H, m,sugar), 4.0 (3H, m, sugar+H3), 4.45 (1H, m), 6.15 (1H, m, sugar), 7.1(1H, 2×s) ppm

δ_(p)(121 MHz; D₂O/H₂O) −10.83 (d, γ-P), -11.46 (d, α-P) and −23.19(t,β-P)

EXAMPLE 10 Synthesis of tritiated6-(2-deoxy-β-D-ribofuranosyl)-324-dihydro-3(R,S)-butyl-8H-pyrimido[4.5c][1,2]oxazin-7-one(10.1)

A solution of6-(2-deoxy-β-D-ribofuranosyl)-3,4-dihydro-3(R,S)-but-5-enyl-8H-pyrimido[4,c][1,2]oxazin-7-one5′-triphosphate (22 mg) in methanol (1.5 ml) was stirred with 10%Pd/C (4mg) and tritium gas (5Ci) for 20 minutes, after which time 1 Ci oftritium gas had been consumed. The catalyst was filtered off using aMilex-SR filter, and labile tritium was removed by repeated evaporationsfrom methanol. The final residue was dissolved in methanol (20 ml). Theyield was 648 mCi. One ml of the above solution was taken to dryness anddissolved in 10 mM aqueous tris buffer pH8 to give a radioactiveconcentration of 8 mCi/ml.

EXAMPLE 11 Primer Extension Assay

In order to test whether the analogue triphosphates were accepted by exominus Klenow polymerase as substrates, primer extension reactions werecarried out with the following templates:

a) 3′ ACGTACACGACCTCTACCTTGCTA 5′

b) 3′ ACGTACACGACCTCTGAACTAGTC 5′

Primer complementary to the sequence underlined was 5′ end labelled with[γ³³P] ATP and T4 polynucleotide kinase. Reactions were boiled for 5minutes after labelling to remove any PNK activity.

For each extension reaction, 1 picomole of ³³P 5′end-labelled primer washybridised with 2 pmole of template in 10 μl ×2 Klenow buffer. Theprimer and template solution was heated at 75° C. for 3 minutes, thenallowed to cool slowly to 30° C. over at least 30 minutes. The solutionwas diluted twice by the addition of 5U exonuclease minus Klenow enzyme(Amersham), 2 mU inorganic pyrophosphatase (Amersham) with 40 μManalogue nucleoside triphosphate and/or 4 μM TTPαS or 250 μM dATP anddGTP. Reactions were incubated at 37° C. for 30 minutes, then stopped bythe addition of formamide stop solution. Reaction products wereseparated on a 19% polyacrylamide 7M urea gel and sized by comparisonwith a ³³p labelled 8 to 32 base oligonucleotide ladder after exposureto Biomax autoradiography film or a phosphor screen (Stormphosphorimager, Molecular Dynamics).

Usng the first template, single base extension was seen as expected withTTP; controls in the absence of added 17P showed no extension.Similarly, the triphosphates of the methyl P analogue (2.4), the butenylP analogue (4.4), the dinitrophenol-labelled P analogue (3.6), thebenzyl hydrazino P analogue (7.3) and the tritiated analogue (10.1) werelo efficiently incorporated. This is in agreement with publishedobservations that the triphosphate of nucleoside analogue P (dPTP) is agood substrate for Taq DNA polymerase.

Using the second template, single base extension was seen as expectedwith dCTP; controls in the absence of added dCTP showed no extension.Similarly, the triphosphate of the methyl P analogue (2.4), the butenylP analogue (4.4) and the dinitrophenol-labelled analogue (3.6) wereefficiently incorporated. Addition of both purines allowe the extensionof the primer to full length products in the presence of the analogueand the added pyrimidine.

EXAMPLE 12 Oligonucleotide Tailing with Terminal DeoxynucleotidylTransferase

In order to test the ability of the analogue triphosphates to beaccepted by terminal deoxynucleotidyl transferase as a substrate, anoligonucleotide tailing reaction was performed.

A 15 mer primer (sequence: 5′ TGC ATG TGC TGG AGA 3′) and 8 to 32 baseoligonucleotide markers were 5′ end labelled with [γ33p] ATP and T4polynucleotide kinase. Reactions were boiled for 5 minutes afterlabelling to remove any PNK activity. Four picomoles of the labelledprimer, 25 U terminal deoxynucleotidyl transferase and 32 μM dNTP oranalogue triphosphate were incubated in 25 μl 1 OOmM cacodylate bufferpH7.2, 2 mM CoCl₂ and 0.2 mM 2-mercaptoethanol for 90 minutes at 37° C.The reactions were stopped by the addition of formamide stop solutionand the reaction products run on a 19% polyacrylamide 7M urea gel withthe labelled markers. Autoradiography using Biomax film was carried outon the dry gel.

The results showed that of the native bases, dATP and TTP produced thelongest tails, followed by dCTP and then dGTP. While dPTP itself was thebest of the analogue substrates for TdT, appearing to incorporate atleast as well as dATP based on migration of the product on the gel, thetriphosphate of the 3-methyl analogue of P (compound 2.4) was also agood substrate, the tailing products running between those producedusing dATP or TTP and dCTP. The triphosphates of 3-butenyl-P (4.4) andbenzyl hydrazino P (7.3) were also substrates though the products wererather small.

In a separate experiment, it was shown that both thefluorescein-labelled compound (8.5) and the amidocaproate-labelledcompound (8.4) produced tails, the tails being of length 16 to 20 basesand 4 to 5 bases respectively.

EXAMPLE 13 Detection of DNA by Means of Antibodies Directed Against PBase

Antibodies were raised against P base so that DNA containing P could bedetected. In order to conjugate P base to a protein carrier it wasnecessary to add a linker and functional group at the 1 position (whichis normally occupied by a sugar in the nucleoside).

Synthesis of1-(4-carboxybutyl)-3,4-dihydro-8H-pyrimido-[4,5-c][1,2]oxazine-7-onePreparation of 5-(2-hydroxyethyl)uracil (13.1)

5-(2-Hydroxyethyl)uracil was prepared following the method of Lin andBrown, Methods in Molecular Biology vol.26, pages 187 to 206 (1994), ed.S. Agrawal, Humana Press Inc.

Preparation of 1-(4-methylcarboxybutyl)-5-(2-hydroxyethyl)uracil(13.2)

5-(2-Hydroxyethyl)uracil (13.1) (5.38 g, 19.9 mmol) was suspended in1,1,1,3,3,3-hexamethyldisilazane (27 ml) and chlorotrimethylsilane (4ml) under a nitrogen atmosphere. The mixture was refluxed for 2 hours.The reaction mixture was cooled to ambient temperature and then reducedto a yellow oil by rotary evaporation. The oil was redissolved inanhydrous toluene (5 ml) and again reduced to an oil.

The oil was dissolved in anhydrous acetonitrile (20 ml) andmethyl-5-bromovalerate (13 ml, 91 mmol) was added. This reaction washeated at reflux under an atmosphere of argon for 18 hours. The mixturewas then cooled and reduced to an oil by rotary evaporation. The productwas purified by liquid chromatography on silica gel eluting with astepped gradient of chloroform and methanol. The product was isolatedand recrystallized from petroleum ether to yield1-(4-methylcarboxybutyl)-5-(2-hydroxyethyl)uracil (13.2) (6.63 g,71.2%), MP 105° C. The structure was confirmed by mass spectroscopy andproton NMR.

Preparation of 1-(4-methylcarboxybutyl)-5-(2-phthalimido-oxyethyl)uracil(13.3)

1-(4-Methylcarboxybutyl)-5-(2-hydroxyethyl)uracil (13.2) no (6.213 g. 23mmol) was dissolved in anhydrous tetrahydrofuran. N-Hydroxyphthalimide(7.505 g, 46 mmol) and triphenylphosphine (12.046 g, 46 mmol) were addedand the solution stirred at ambient temperature. Diethylazodicarboxylate(9.95 g, 57 mmol) was added in 0.5 ml aliquots over 10 minutes, thereaction was then stirred for a further hour. The product wasrecrystallized twice from chloroform : diethyl ether mixtures to yield1-(4-methylcarboxybutyl)-5-(2-phthalimidooxyethyl)uracil (13.3) (8.77 g,91.8%). The structure was confirmed by mass spectroscopy and proton NMR.

Preparation of1-(4-methylcarboxybutyl)-5-(2-phthalimido-oxyethyl)4-triazolopyrimidine(13.4)

1,2,4-Triazole (11.843 g, 171 mmol) was dissolved in anhydrousacetonitrile (200 ml) and stirred at 0° C. Phosphorus oxychloride (3.9ml, 42 mmol) was added dropwise followed by triethylamine (31 ml, 225mmol). Stirring was continued at 0C for 1 hour.1-(4-Methylcarboxybuty1)-5-(2-phthalimido-oxyethyl)uracil (3) (4.16 g,10 mmol) dissolved in anhydrous tetrahydrofuran (50 ml) and anhydrousacetonitrile (80 ml) was added slowly to the above stirred solution. Themixture was heated at 50° C. for 2.5 hours and then cooled to 0° C. toyield a white precipitate. The product was further purified by columnchromatography on silica gel, eluting with chloroform : acetone (7:3).This produced1-(4-methylcarboxybutyl)-5-(2-phthalimido-oxyethyl)-4-triazolouracil(13.4) as an oil (3.43 g, 73.4%). The structure was confirmed by massspectroscopy and proton NMR.

Preparation of 1-(4-methylcarboxybutyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]oxazine-7-one (13.5)

1-(4-Methylcarboxybutyl)-5-(2-phthalimido-oxyethyl)-4-triazolopyrimidine(4) (0.49 g, 1.05 mmol) was dissolved in anhydrous 3) dioxan (30 ml).Anhydrous dioxan saturated with ammonia (70 ml) was added and thereaction mixture was stirred for 24 hours. The product was purified bythin-layer chromatography on silica gel GF plate eluted with chloroform: ethanol (95:5) (0.21 g, 74.9%). The structure was confirmed by massspectroscopy.

Preparation of1-(4-carboxybutyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]oxazine-7-one,ammonium salt (13.6)

1-(4-Methylcarboxybutyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]oxazine-7-one (13.5) (0.42 g, 1.57 mmol) was dissolved in 1Maqueous sodium hydroxide solution (10 ml) and stirred for 2 hours. Themixture was applied to an ion exchange column of Dowex 50 WX8-200. Thiswas eluted with water and the product recovered using 2M aqueous ammoniasolution. The solution volume was reduced and then freeze-dried to yielda white solid (0.24 g, 60.4%). The product appeared as a single spotwhen analysed in the following thin-layer chromatography systems: Silicagel eluted with chloroform:methanol (7:3) and butan-1-ol: water, glacialacetic acid (12:5:3); Silica gel impregnated with ODS eluted withmethanol:water:glacial acetic acid (80:20:1); Cellulose eluted withbutan-1-ol:water:glacial acetic acid (12:5:3). The structure wasconfirmed by mass spectroscopy and proton NMR.

Preparation of P base KLH conjugate (13.8)

1-(4-Carboxybutyl)-3,4-dihydro-8H-pyrimido[4,5-c][1,2]oxazine-7-one (6)(41.6 mg, 0.164 mmol) was dissolved in dimethylsulphoxide (2 ml).N-Hydroxysuccinimide (26.5 mg, 0.23 mmol) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (65.4 mg, 0.341 mmol) wereadded and the mixture was stirred at ambient temperature for 26 hours.The dimethylsulphoxide was removed under vacuum. The residue waspurified by column chromatography on silica gel and eluting withacetone.

The product (13.7) was isolated by removing the solvent by rotaryevaporation.

The N-succinimidyl ester (13.7) was dissolved in DMF (1 ml) and the KLHsuspension (90 mg protein) dissolved in pyridine (0.5 ml) and water (4.5ml) was added. The mixture was stirred under an atmosphere of nitrogenfor 4 hours. The product was dialysed with water for 5 days. Theconjugate (13.8) was isolated by freeze-drying to yield 59 mg. This wassubmitted to Polyclonal Antibodies Ltd for antiserum production.

Antiserum Production

Three sheep were immunised with the KLH conjugate. The primaryimmunisation was carried out with the conjugate formulated in Freundscomplete adjuvant. Subsequent reimmunisations were carried out with theconjugate formulated in Freunds incomplete adjuvant at 4 weeklyintervals. Blood samples were taken 2 weeks after each reimmunisationand serum prepared.

Testing of Antisera

Antisera were tested against dot blots of P-labelled oligonucleotides (i.e. P itself being the label) on nylon membrane using a second antibodyconjugated to horseradish peroxidase with ECL substrate for detection.The response of pre-immune serum taken from each animal was comparedwith serum taken from the first and second bleeds.

One microlitre aliquots containing 10, 5, 1, 0.5, 0.1, 0.05 and 0.01pmole of P-labelled oligonucleotide (sequence: 5′ PPP GTC ACG AC 3′)diluted in water were dotted onto Hybond N+ membrane. A one microlitrealiquot of control oligonucleotide (sequence: 5′ TGC TGG AGA 3′) dilutedin water was also applied. Blots prepared in this way were baked at 80°C. for 90 minutes to fix the DNA. After baking, a one microlitre aliquotof a 1:1000 dilution of pre-immune serum was dotted onto each blot togive a positive control.

The blots were incubated for 60 minutes at room temperature with shakingin Liquid Block (Amersham) diluted 1:10 with 10 mM phosphate bufferedsaline (PBS). Individual blots were then incubated for 60 minutes withshaking in 1:1000, 1:10000 or 1:50000 dilutions of each serum sample in0.5% bovine serum albumin (BSA) solution in PBS. They were then washed 3times for 10 minutes each wash with shaking in PBS containing 0.3% Tween20. The blots were then incubated for 60 minutes with shaking inhorseradish-peroxidase conjugated affinity-purified donkey anti-sheepIgG, H+L (Jackson lmmunoResearch Labs Inc) diluted 1:25000 in 0.5% BSAin PBS, then washed three times for 10 minutes each wash with shaking inPBS containing 0.3% Tween 20. They were then incubated in ECL detectionreagent (Amersham) for 1 to 2 minutes and then exposed to Biomax film(Amersham) for 2 minutes and 5 minutes.

Positive signals were seen from antisera obtained after immunisation ofall three sheep at all dilutions. A maximum sensitivity of 0.1 picomoleP-labelled oligonucleotide was obtained with the 1:1000 antiserumdilution. Some background signal was seen with the unlabelledoligonucleotide at the 1:1000 dilution of all the serum samples,including the preimmune serum, but this was not seen at the 1:10000 or1:50000 dilutions. A strong signal was obtained from the positivecontrol.

This experiment demonstrates the potential for detecting or capturingoligonucleotide or other probes tailed or otherwise labelled withnucleotide analogues.

EXAMPLE 14 Detection of M13 on Dot Blots wing 3′end labelled probes

Oligonucleotide probes labelled with fluorescein- ordinitrophenol-containing nucleotide analogues were detected afterhybridisation to target DNA immobilised on a membrane.

Probe Preparation

Six microlitres (12 picomoles) −40 forward sequencing primer 23 mer(sequence 5′GTTTTCCCAGTCACGACG1TGTA-3′) was mixed with 10 μl×5 reactionbuffer 500 mM sodium cacodylate pH 7.2, 10 mM CoCl₂, 1 mM2-mercaptoethanol), 50 units terminal deoxynucleotidyl transferase, 15μl 800 μM fluorescein labelled compound (8.5) or the dinitrophenollabelled compound (9.3) and water added to give a final volume of 50 μl.Both reactions were incubated at 37° C. for 90 minutes. The reactionswere then boiled for 5 minutes to remove any remaining terminaldeoxynucleotidyl transferase activity.

Blot Preparation

Dot blots of M13 single-stranded DNA were prepared on Hybond N+membrane. Eight dots were applied to each membrane containing 1200, 240,120, 48, 36, 24, 12 and 6 picograms corresponding to 500, 100, 50, 20,15, 10, 5 and 2.5 attomoles of target respectively. The dots were fixedto the membrane by baking in an oven at 80° C. for 90 minutes.

Hybridisation Method

Duplicate dot blots were pre-hybridised in 5 mi hybridisation buffer(0.5% dextran sulphate, 0.1% sodium dodecyl sulphate (SDS), 1:20 liquidblock (Amersham Life Science) and x5 sodium chloride sodium citratebuffer (SSC)) for 30 minutes at 42° C. Twenty-five microlitres (˜50 ng)of the probes prepared above was added to each hybridisation to give aprobe concentration of 10 ng/ml. The blots were placed in a 42° C.shaking waterbath for 60 minutes.

The blots were then washed in 5 ml×5 SSC/0.1% SDS at room temperaturefor 5 minutes. This wash was repeated with a further 5 ml×SSC/0.1% SDSat room temperature for 5 minutes.

The blots were then washed in 5 ml×l SSC/0.1% SDS at 42° C. for 15minutes. This wash was repeated with a further 5 ml×1 SSC/0.1% SDS at42° C. for 15 minutes.

The blots were then transferred to 1:10 dilution of liquid block,Amersham, diluted in TBS buffer (0.3M sodium chloride 0.1M Trizma basepH 7.5) and placed on an orbital shaker for 30 minutes.

For the detection of DNP-containing probes, blots were transferred to 5ml 0.5% w/v BSA in TBS buffer pH 7.5. To this was added 5 μl anti-DNPhorseradish peroxidase conjugated antibody (giving a 1:1000 dilution ofthe antibody).

For the detection of fluorescein-containing probes blots weretransferred to 5 ml 0.5% w/v BSA in TBS buffer pH 9.5. To this was added5 μl anti-fluorescein alkaline phosphatase conjugated antibody (gives a1:1000 dilution of the antibody).

Both sets of blots were them transferred to an orbital shaker for 45minutes.

The blots were drained and transferred to either 25 ml 0. 1% vv Tween-20in TBS pH 7.5 (DNP-labelled probe) or 25 ml 0.1% v/v Tween-20 TBS pH 9.5(fluorescein-labelled probe) and placed on the orbital shaker for 15minutes. This wash step was repeated, three times in total, with afurther 5 ml 0.1% v/v Tween-20 TBS buffer at the relevant pH.

Detection of alkaline phosphatase (fluorescein-labelled probe)

The blots were drained and transferred to Saran Wrap. 0.5 ml CDP-Star™reagent was applied to each blot and left at room temperature for 5minutes. The blots were drained then wrapped in Saran Wrap and exposedto autoradiography film for 30 minutes. On the developed film it waspossible to see all the dots from the 500 to the 2.5 attomole dot.

Detection of Horseradish Peroxidase (DNP-Labelled Probe)

The blots were drained and transferred to Saran Wrap. A 0.5 ml aliquotof ECL detection reagent (Amersham) (made by mixing solution 1 and 2 inequal amounts) was applied to each blot and left at room temperature for2 minutes. The blots were drained then wrapped in Saran Wrap and exposedto autoradiography film for 10 minutes. On the developed film it waspossible to see the 500 and 100 attomole dots. Greater sensitivity canbe obtained by optimisation of the antibody concentration to reducebackground.

What is claimed is:
 1. A nucleoside analogue of the formula

wherein X is O, S, Se, SO, CO or N—R¹⁰, the dotted line represents anoptional link between R⁶ and R¹⁰, R¹, R², R³ and R⁴ are the same ordifferent and each is H, OH, F, NH₂, N₃, O-hydrocarbyl, or a reportermoiety, R⁵ is OH or mono-, di- or tri-phosphate or -thiophosphate orcorresponding boranophosphate, or one of R² and R⁵ is selected from thegroup consisting of a phosphoramidite, H-phosphonate, methylphosphonate,and phosphorothioate for incorporation in a polynucleotide chain, Z isO, or S, and R⁶, R⁷, R⁸, R⁹ and R are the same or different and each isH or alkyl or aryl or a reporter moiety, n is 0 or 1, provided that atleast one reporter moiety is present on a member selected from the groupconsisting of R¹ to R⁴ and R⁶ to R¹⁰, wherein a reporter moietycomprises a linker group, together with a signal moiety or a solidsurface, or a reactive group for attachment of a signal moiety or asolid surface to the nucleoside analogue.
 2. A nucleoside analogue asclaimed in claim 1, wherein X is O and Z is O.
 3. A nucleoside analogueas claimed in claim 1, wherein R⁵ is triphosphate.
 4. A nucleosideanalogue as claimed in claim 1, wherein R⁶ or R¹⁰ comprises a reportermoiety.
 5. A nucleoside analogue as claimed in claim 1, wherein thereporter moiety comprises a signal moiety and a linker group.
 6. Anucleoside analogue as claimed in claim 1, wherein the linker group is achain of up to 30 carbon, nitrogen, oxygen and sulphur atoms, rigid orflexible, unsaturated or saturated.
 7. A nucleoside analogue as claimedin claim 1, wherein the reactive group is NH₂, OH, COOH, CONH₂, or SH.8. A polynucleotide chain comprising at least one residue of anucleoside analogue according to claim
 1. 9. A nucleoside analogue asclaimed in claim 8, wherein one of R² and R⁵ is phosphoramidite orH-phosphonate.
 10. A polynucleotide chain as claimed in claim 8, whereina signal moiety has been introduced into the incorporated nucleosideanalogue residue.
 11. A method of detecting a nucleic acid whichcontains a residue of a nucleoside analogue of the formula

wherein X is O, S, Se, SO, CO or N—R¹⁰, the dotted line represents anoptional link between R⁶ and R¹⁰, R¹, R², R³ and R⁴ are the same ordifferent and each is H, OH, F, NH₂, N₃, O-hydrocarbyl, or a reportermoiety, R⁵ is OH or mono-, di- or tri-phosphate or -thiophosphate orcorresponding boranophosphate, or one of R² and R⁵ is a phosphoramiditeor other group for incorporation in a polynucleotide chain, Z is O, orS, and R⁶, R⁷, R⁸, R⁹ and R¹⁰ are the same or different and each is H oralkyl or aryl or a reporter moiety, n is 0 or 1, which method comprisesthe steps of: (a) combining said nucleic acid with an antibody whichbinds to said analogue; and (b) detecting the presence of said nucleicacid.